Project Summary Molecular fingerprinting of chromosomal deletions and DNA mutations is increasingly important in the clinical management of patients with glioblastoma (GBM) ? the most common and deadly primary adult brain tumor. While clinical analyses of tumor DNA have to this point relied on neurosurgical specimens, non-invasive detection through liquid biopsy of circulating tumor DNA (ctDNA) has the potential to revolutionize the detection and monitoring of these aggressive, heterogeneous, and treatment resistant neoplasms. Although several groups have detected ctDNA in patients with non-central nervous system (CNS) tumors, the protected location of brain tumors within the CNS likely limits the passage of molecules like ctDNA through the blood brain barrier (BBB). A new non-invasive technology to disrupt the BBB is magnetic resonance imaging-guided focused ultrasound (MRgFUS). MRgFUS can safely deliver ultrasound energy across the intact skull with high precision and accuracy, and is FDA-approved for the treatment of certain neurodegenerative conditions using high energy thermal lesioning. MRgFUS-mediated BBB disruption is accomplished with relatively low energy settings which are used to oscillate circulating microbubbles, perturbing and temporarily disrupting the BBB. The project team is leading the US clinical trials investigating MRgFUS-mediated BBB disruption in brain cancer patients, and have been pioneers in the sensitive and specific detection of ctDNA in the setting of solid tumors. Accordingly, we propose investigating MRgFUS to enable liquid biopsy of brain cancer through extra-CNS release of tumor DNA. Our overall hypothesis is that BBB disruption localized to specific brain tumor regions using MRgFUS will release tumor region-specific DNA into the circulation and this ctDNA will be detectable using our highly sensitive DNA sequencing technology. We further predict that the ultrasound settings can be further tuned and optimized to maintain safety and increase release of ctDNA to improve detection/diagnostic yield. We will test this hypothesis in two specific aims: In Aim 1, we will investigate the dynamics of FUS-triggered release of glioma DNA into the blood stream in advanced, faithful pre-clinical GBM rat models and determine the optimal settings and timing of tumor DNA release while maintaining safety. In Aim 2, we propose to assess the presence of tumor-specific DNA in the blood of brain tumor patients who will be already enrolled in MRgFUS-mediated BBBD clinical trials lead by our team. Successful completion of the work with establish the potential for MRgFUS enabled, non-invasive biopsy of GBM, which would radically advance the diagnosis and monitoring of human brain cancer.